Sep 08, 2020
Version 1
High-throughput SARS-CoV-2 RNA extraction from nasopharyngeal swabs using repurposed 3D printers. V.1
- 1Institut Pasteur du Cambodge
- Coronavirus Method Development Community
- XPRIZE Rapid Covid Testing
Protocol Citation: Koen Vandelannoote 2020. High-throughput SARS-CoV-2 RNA extraction from nasopharyngeal swabs using repurposed 3D printers.. protocols.io https://dx.doi.org/10.17504/protocols.io.bkynkxve
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 07, 2020
Last Modified: September 08, 2020
Protocol Integer ID: 41710
Keywords: Ender VX-500, RNA extraction, 3D printer, SARS-CoV-2, nasopharyngeal swabs, COVID-19
Abstract
Here, we describe a semi-automated workflow for paramagnetic bead-based extraction of SARS-CoV-2 RNA from human upper respiratory samples that uses low-cost repurposed Creality3D Ender-3 3D-printers and readily accessible reagents. The protocol described here is for the use of four instruments in parallel, each processing 24 samples, 96 samples per batch.
Materials
REAGENTS:
- Lysis buffer (6 Molarity (M) Guanidinium thiocyanate , 150 millimolar (mM) Tris-Cl 8 , 30 millimolar (mM) EDTA 8 , 90 millimolar (mM) DTT , 3% (v/v) Triton X-100).
- SPRI bead mix prepared for RNA binding as described in https://openwetware.org/wiki/SPRI_bead_mix
Note
This SPRI bead mix is a drop-in substitute for RNAClean XP beads (Beckman Coulter) at about 1/100 of the cost.
- 100% Ethanol
- 80% Ethanol solution
- Nuclease free water (NFW)
PLASTIC CONSUMABLES:
- 96-well plates with V-shaped wells (Greiner – Ref. 651201)
Note
It is highly recommended to work with the specified 96-well plates. The use of alternative plates requires careful modifications to the Ender VX-500 "print protocol”.
- 8-tube PCR strips (SSIbio - Ref. 3247-00)
Note
It is highly recommended to work with the specified strip tubes. The use of alternative strips requires careful modifications to the Ender VX-500 "print protocol”.
- 96 deep-well plates (Axygen product number P-DW-11-C-S)
Safety warnings
Nasopharyngeal swab samples that may contain respiratory viruses should be handled in a Class II BSC. Standard PC2 laboratory personal protective equipment is required. This includes a long sleeved laboratory gown, gloves and safety glasses. Additionally, a face mask should be worn.
The lysis buffer contains guanidine thiocyanate which may produce hazardous gases (including hydrogen cyanide and chlorine) when combined with bleach (sodium hypochlorite) and/or strong acids.
Ethanol is a highly flammable liquid.
Before start
This protocol provides directions for the use of a 3D Printer RNA extraction apparatus we named the "Ender VX-500". The platform essentially consists of a Creality3D Ender-3 printer repurposed by installing three different custom designed interlocking attachments on the printer's extruder mounting mechanism.
These models can be obtained from https://www.thingiverse.com/koen_vdl/designs
A 3D print “job” can be executed on the printers which will emulate the movements required to make the magnetic combs on the mounted attachments run a paramagnetic bead-based RNA extraction protocol.
Here we describe how we can efficiently process 91 nasopharyngeal swab samples in parallel on 4 Ender VX-500 machines. The following is a graphical overview of the extraction precidure.
Pre-process sample information
Pre-process sample information
Scan the barcodes of 91 nasopharyngeal swabs using a barcode scanner and the "20200908_Ender_VX-500_extraction_template.xlsx" spreadsheet.
- Populate the “SampleID” column of the “Sample log” sheet
- Randomly include a negative extraction control for every set of 24 swabs in the sheet.
- Randomly include a PCR no-template-control (NTC) control in the sheet.
Note
The spreadsheet will automatically parse 5 plate dispensing charts that can be printed for convenience:
- 4 charts represent the plate layouts of the 4 Ender VX-500 extraction runs (of 24 samples each).
- 1 chart represents the plate layout of the 96-well plate on which the eluates of the 4 extraction runs will be combined.
Note
The "20200908_Ender_VX-500_extraction_template.xlsx" spreadsheet can be found as an attachement under the abstract tab.
Pre-dispense the samples into a deep-well 96 well plate
Pre-dispense the samples into a deep-well 96 well plate
Transfer 100 uL of swab transport medium into a deep-well 96 well plate in a Class II BSC using the plate layout charts as a guide.
Prepare four Ender VX-500 machines for extraction
Prepare four Ender VX-500 machines for extraction
Attach three 8-tube PCR strips onto the “Tube holder”.
Note
- The caps of the strip fit tightly into the openings on the “Tube holder”.
- Only hold the strips by the caps and make sure not to touch the outside of the tubes.
Seat the “Tube holder” into the “Adaptor”.
Lock the “Tube holder” in place by inserting the provided Allan hex wrench. This will ensure the “Tube holder” is always inserted in the same position.
Place three “Magnetic bars” in the openings of the “Tube holder”.
Prepare four 96-well plates for extraction
Prepare four 96-well plates for extraction
Add 18.5 µL Lysis buffer to columns 1, 5 and 9 of a 96-well plates with V-shaped wells using a multichannel pipette.
Note
Resuspend any salted out guanidine thiocyanate crystals before dispensing the buffer.
Incubate 65 °C for 00:10:00
Add 80 µL NFW to columns 4, 8 and 12 using a multichannel pipette.
Add 150 µL 80 % (v/v) Ethanol to columns 2, 3, 6, 7, 10 and 11 using a multichannel pipette.
Note
- The ethanol concentration will quickly lower in the air flow of the BSC.
- Always use a freshly made up Ethanol solution.
Transfer / pipet-mix37 µL of nasopharyngeal swab transport medium from the deep-well plate into columns 1, 5 and 9 using using a multichannel pipette and the relevant plate dispensing chart
Incubate the plate Room temperature for 00:01:00 .
Transfer / pipet-mix 30 µL SPRI bead mix into columns 1, 5 and 9 using a multichannel pipette.
Note
Shake the SPRI bead mix before use (the beads settle quickly).
Transfer / pipet-mix 85.5 µL 100 % (v/v) Ethanol to columns 1, 5 and 9 using a multichannel pipette.
Incubate the plate Room temperature for 00:05:00 .
Note
The prepared plates need to be extracted on the Ender VX-500 machines immediately after this incubation step
Extract 4 plates on 4 Ender VX-500 machines
Extract 4 plates on 4 Ender VX-500 machines
Insert the four 96-well plates onto four Ender VX-500 machines prepared for extraction
Note
Ensure the microplates are correctly oriented by checking the position of the A1 marker.
Power on the Ender VX-500 machines.
Use the knob on the Ender VX-500 to start the extraction by selecting “Print from TF” and selecting “RNA_ex_v10.gcode”.
Note
The "RNA_ex_v10.gcode" print job which emulates the RNA extraction can be found as an attachment under the abstract tab.
The Ender VX-500 will:
- Collect bead-RNA complex from the lysate (00:05:00 )
- Wash 1: vortex (00:01:00 ) & collect bead-RNA complex (00:01:30 )
- Wash 2: vortex (00:01:00 ) & collect bead-RNA complex (00:01:30 )
- Air dry (00:05:00 )
- Elute: vortex (00:01:00 ) & collect beads (00:01:00 )
- Raise the magnetic bars out of the eluates
Note
After00:20:00 the machine will beep to indicate the run completed successfully.
Remove the 96-well plates from the Ender VX-500 machines.
Power off the Ender VX-500 machines.
Place the 96-well plates on a 96 well magnetic rack and use a multichannel pipette to transfer 50 µL of eluate onto a fresh 96-well plate that will combine the eluates of the 4 extraction runs.
Note
30 µL of eluate is sacrificed to prevent any bead SPRI bead carryover.
Machine clean-up
Machine clean-up
Carefully remove the "Tube holder" from the EnderVX-500 “Adaptor ”.
Use paper tissues to release the three 8-tube PCR strips from the "Tube holder" and decontaminate the attachment in a bath containing a 1% sodium hypochlorite solution for min 00:10:00 .